In Egypt, seven disciplines of FE research were undertaken: insect succession (23 studies), developmental studies (9), entomotoxicology (8), insect identification (9), electrophysiological study (2), biochemical study (1), and a human case study (3). Throughout these studies, Hegazi et al. (1991) conducted the first research in Matrouh (Western desert), followed by EL-Kady et al. (1994a, b) in Alexandria. El-Shazly et al. (1995) conducted research in Giza, and AbouZied et al. (2003) began their research in Cairo. The research continued in further locations, with the most recent being handled in Alexandria (El-Samad et al., 2020), Cairo (Abd El-Gawad et al., 2020), Giza (AbouZied et al., 2020), and Sharqia (Farag et al., 2021).
Carcass decomposition, insect distribution, and succession studies
Over time, the carcass decomposed into well-known decaying stages (Henssge et al., 1995), drawing flies, beetles, ants, and a range of more arthropods (Catts & Haskell, 1990). Temperature, humidity, clothing, burning, and food availability are all elements that impact carrion decay and SPs in each location (Campobasso et al., 2001). In addition, the PMI may also be estimated by examining arthropod SPs and stages of body decay (Smith 1986; Voss et al., 2011; Morris & Dadour, 2015). In Egypt, research on corpse decomposition and SPs were carried out in eleven governorates that may represent various settings.
In the Western desert of Matrouh governorate, Hegazi et al. (1991) studied the monthly SPs of insects on the flesh of a mature snail and a small carp in two habitats (above and below the ground). Nine insect families were discovered, with 19 insect species belonging to the three orders of flies, beetles, and ants. Insects appeared to like fish baits somewhat more than snail baits. During the study period, flies were the most frequent insects on both baits, with the Calliphorid family being the most common. Diptera seemed to consume aboveground carrion, whereas Histerids and Dermestids beetles were found both above and below the ground surface. According to this study, the SPs of insects differed dependent on the carcass type and habitat.
Rabbits were employed as experimental animals in six studies. Seasonal fluctuations, as well as the reasons of mortality and the stage of decomposition (El-Kady et al., 1994a, EL-Kady et al., 1994b), have been found to influence the decaying process and SPs (Tantawi et al., 1996). Tantawi et al. (1998) also looked at the fly larvae SPs on rabbit carcasses subjected at the same time and in the same place from three groups, blowflies, flesh flies, and house flies. The other two experiments looked at the impacts of propoxur (Abdel Ghaffar et al., 2008) and amitriptyline (Abdel Ghaffar et al., 2008). (El-Samad & Youssef, 2016). The presence of such chemicals impacted the duration of decay stages, SPs, and the rate at which their immature stages grew, according to the researchers. Researchers in Alexandria observed that insect SPs were impacted by mortality reasons, seasonal changes, and the existence of chemicals.
Gabre (2002) and Gabre and Abou Zied (2003) conducted a seasonal field research using four bait traps (fish, bovine-lung, grapes, and molasses) to characterize the abundance levels, pattern, and sex ratio of carrion flies. The most abundant flies found during the trial were Lucilia cuprina Wiedemann, Chrysomya megacephala Fabricius, and Musca domestica Linnaeus. Furthermore, the almost identical number of male and female flies captured on grape and molasses baits might represent the real male-female population balance. Female flies were also found in far greater numbers on fish and bovine-lung baits than on grape or molasses baits. Lastly, the number, distribution, and sex ratio of carrion flies were all impacted by the bait type, according to the two studies in Suez.
In the Governorate, two studies were included. Abd EL-Bar and Sawaby (2011) explored the role of pirimiphos-methyl, an organophosphate pesticide, on the process of decay and insect attractiveness. They observed that whereas the bodies of control rabbits decomposed quickly, the carcasses of treated rabbits did not, even after 40 days. The same group of insects invaded the guinea pig and rabbit carcasses, with Chrysomya albiceps Wiedemann being the first to arrive. On the treated carcasses, there were less blowfly larvae, which might explain the unusual decaying delay. Formicidae were noticed in all dead bodies, particularly in the early stages of decay. Ibrahim et al. (2013) discovered that guinea pig corpses decayed quickly in the fall and summer, that insect attraction followed a predictable seasonal pattern, and that the insect colony grew in richness as the decay continued. Seasonal and decaying stage differences, or the existence of chemicals, all influence the insect exposed to carcasses, according to the two experiments in El-Qalyubiya.
Aly et al. (2013) conducted a research in Ismailia, where they examined SPs and identified attracted species on rabbit and rat remains during the summer. Four fly families, two beetle families, and one ant species (Monomorium minimum Buckley) were among the insects gathered. Finally, the research showed that the decay rate and the number of visitor species varied depending on the decay stage.
In Cairo, six studies were conducted, four of which looked at the effects of seasonal and habitat change and two of which looked at the effects of chemicals. Using rabbits and dogs as animal models, Zeariy et al. (2015, 2018) and Zeariya and Kabadaia (2019a, b) examined the different influences of carcass decomposing stages, carcass type, and habitats (indoor and outdoor) on insect diversity and SPs. According to the studies, the blowfly, Ch. albiceps, was the first fly driven to the initial days of decaying. Likewise, the insect variety on the two carcasses looked to be rising outdoors and decreasing indoors, with larger numbers seen on dog carcasses than rabbit carcasses. Also impacted by the decaying stage are the amount and variety of insects. In particular, the warmer seasons had the shortest larval and pupal lengths, whereas the cold seasons saw the longest larval and pupal lengths.
Zinc phosphide-intoxicated (Abd EL-Bar et al., 2016) and warfarin (Abd El-Gawad et al., 2019) chemicals have been studied for their influence on SPs and decomposing processes. Control carrions decomposed quicker than treated corpses, according to Abd EL-Bar et al. (2016), whereas treated carcasses decomposed faster than controls, according to Abd El-Gawad et al. (2019). In addition, control carcasses attracted more insects than zinc phosphide-intoxicated treated carcasses, but warfarin carcasses attracted much more insects than control bodies. Furthermore, Abd El-Gawad et al. (2019) remarked that the rabbit fur color influences insect attractiveness, with black rabbit remains attracting more adults than other colors.
According to the studies conducted in Cairo, changes in carcass type and habitat impact the sorts of insects involved and the SPs. Additionally, the occurrence of chemicals influenced the decay rate and the attractiveness of insects to carcasses.
The effect of tramadol on rat carcass decomposition, SPs, and rate of growth of Sarcophaga argyrostoma Robineau-Desvoidy was investigated in a study by AbouZied (2016). According to the research, the decomposition of the treated carcass was not substantially quicker than that of control carcasses. Chrysomya albiceps, L. cuprina, and M. domestica were fewer in treated carcasses than control carcasses. Female Sarcophaga spp., in contrast, were more drawn to the treated ones. Sarcophaga argyrostoma maggots collected from treated groups were much longer than that collected from control groups, and maggots fed treated groups pupated 2 days later than maggots fed control groups. As a result, tramadol had no effect on carcass decay, but it did affect the insect appeal to carcasses.
Aly et al. (2017) did a research in Qena that looked at the insects that were associated to rabbit carcasses in both outdoor and indoor settings. They found that, in comparison to winter and fall, the rate of body decomposition was quicker in summer and spring. Outdoor bodies decay more quickly than indoor bodies. Outdoor bodies decay more quickly than indoor bodies. There was a total of 18 species identified, including 5 in the outdoors, 3 in the indoors, and 10 common species among them. Twelve Diptera, three Coleoptera, and three Hymenoptera species were reported. Blowflies and flesh flies were also the earliest to appear on rabbit carcasses. According to the authors, variations in decomposing stages, place, and seasonality impacted insect types and succession tendencies.
Abd El-Aziz and El Shehaby (2019) analyzed the forensic arthropod species that colonizes rat carrions in an aerated habitat throughout the spring season. Consequently, the fauna consists of 13 species of insects from 10 families, with blowflies being the first to arrive, followed by other flies in various stages of decay, and beetles limited to the late stages. Finally, the date of the initial insect appearance and the insect type varies depending on the decay stage.
The effects of clothes on the decomposition and insect attractiveness to rabbit carcasses were investigated by AbouZied et al. (2020). They discovered that clothes considerably slowed the decay rate and that the existence of clothes had no impact on the insect species drawn to the carcass or the SPs recorded.
During the summer season, Farag et al. (2021) looked at the variety and SPs of insects that colonized indoor rat carrion killed by various methods (cervical displacement, electrocution, immersion, or slaughtering). The most prevalent insects discovered were flies and beetles, with blowflies being the first to occupy the carrion and lasting until the start of the dry stage. In addition, neither the SPs nor the number of related insects varied between the control and immersion groups. There was, however, a distinction between the electrocuted and slaughtered groups.
Insect growth and development studies
It is crucial to know which insects are attracted to dead bodies in a specific region, as well as the growth rates of prominent carrion insect larvae, when estimating PMI using insect data (Gennard 2007). According to Tarone and Benoit (2019), the location temperature data is critical for complete entomological investigation since insects have temperature-dependent life cycles. Still, temperature is the extremely considerable piece of data applied to PMI estimation based on the growth rates of insects, as per Byrd and Sutton (2020).
Nine developmental experiments were brought in six governorates, focusing on five flies: four blowflies and one flesh fly. El-Shazly et al. (1995) compared the developing and propagative characteristics of Ch. albiceps and Parasarcophaga argyrostoma, then age-specific survival and productiveness. They noticed that the two flies are on opposite ends of the r-strategist spectrum. Kheirallah et al. (2006, 2007) investigated the temperature effects on the growth of Lucilia sericata Meigen and Ch. albiceps using four different temperatures. According to the studies, Ch. albiceps has a greater growth level and a longer larval development time than L. sericata. Moreover, it was discovered that in pure and mixed cultures of varied densities, Ch. albiceps development was unaffected when L. sericata density grew while Ch. albiceps density stayed constant. Likewise, the sex ratio of both species developing in pure and mixed cultures was unaffected by egg density or temperature, and greater extents resulted in lower adult sizes for both species.
Rashed et al. (2015) used Ch. albiceps to probe the impact of temperature changes on growth rate in distinct seasons. They observed that the total development period of the blowflies was affected by temperature fluctuations and showed substantial seasonal variability. High temperatures promote the growth of the fly, but low temperatures slow it down. Females emerged at higher rates than males, and the female ratio was greater throughout the winter. Abd El-Gawad et al. (2018) also looked at how the rodenticide warfarin influenced the growth and survival of fly maggots (L. sericata, Ch. albiceps, and Ch. megacephala). They discovered that treated larvae grew faster than control larvae, and seasonal temperature fluctuation is a crucial element in the blowflies’ growth time.
Gabre et al. (2005) investigated the Ch. megacephala life cycle in the lab at a temperature of 26 °C, where the two-sex life table assessment offers a thorough explanation of the fly developmental stage differentiation. Ibrahim et al. (2014) also investigated the effect of larval density on the fly population ecology and survival. They observed that there is a link between larval density and survival, that density affects pre-oviposition time but not sex ratio, and that density influences fertility.
AbouZied et al. (2003) used an age-stage, two-sex life table, and different growth rates among individuals to evaluate raw data on L. cuprina life history in a lab setting. The outcomes showed that the L. cuprina population is r-strategist. Also, Elshehaby et al. (2016) looked at the impacts of three various types of feed (liver, beef, and meat) on the number of L. sericata and their growth time. They determined that the development rate of all stages of cultured flies increases when the kind of meat diet changes, flies prefer meat other than liver and beef, and the development characteristics of L. sericata were substantially higher with diet meat than with the other diets examined. As a result, the authors concluded that diet meat may be used to mass rear blowfly larvae for a variety of fundamental and applied research projects.
According to the developmental research, temperature is the most essential element influencing the flies’ life cycle, with a substantial influence on pre-oviposition and incubation periods, egg hatching percentage, female fecundity, and larval growth time.
The precise and rapid classification of insects is a first and essential stage in using insect proof in forensics. According to Tang et al. (2012), traditional morphological insect identification poses a considerable challenge for forensic scientists, but genetic approaches for insect identification are faster and more efficient.
In Egypt, Tantawi and El-Kady (1997) used morphological methods to distinguish third-instar larvae of various flies from blowflies, flesh flies, and house flies, while Sawaby et al. (2016) identified 29 species from 9 families in a taxonomic study on beetles, and Sawaby et al. (2018) used the same characteristics to identify dipterous species.
A few articles, instead, used molecular identification, such as in a master research by El-Hefnawy (2013), data assessment utilizing the RAPD polymerase chain reaction technology proved the third larval instar of Sarcophaga carnaria Linnaeus. Aly (2014), Salem et al. (2015), and Abdel Ghaffar et al. (2018) also used short and long cytochrome oxidase I gene segments (COI) to distinguish between flies.
Furthermore, Elleboudy et al. (2016) used a new method called focus stacking to identify 76 Chrysomya species in Egypt, with Ch. albiceps and Ch. megacephala being the most common species. El-Hefnawy et al. (2020) explored the possible function of genome size measurements in forensics by using flow cytometry to explain the importance and ease of use of genome size estimates in flies (Wohlfahrtia magnifica Schiner and S. argyrostoma) obtained from Aswan and Benha. The study found variations in genome size within and between the juvenile stages of the two species. Finally, the forensic insects were identified using both morphological and molecular methods in the classification experiments.
Entomotoxicology is commonly applied when toxicological tests on bodily tissues are not possible (Verma & Paul, 2013), or when internal organs, blood, or urine are not available for a criminal probe, entomotoxicology is frequently used (Sankhla et al., 2017). Several studies in Egypt employed it to detect the existence of chemicals in insect larvae and body tissues, as well as their effects on larval growth.
Tantawi et al. (2001) utilized a radioimmunoassay technique to measure the level of morphine in the 3rd instar larvae of blowflies and flesh flies attracted to rabbit carcasses left outdoors. El-Samad et al. (2011) employed high-performing liquid chromatography with a UV-visible spectrophotometric detector to identify tramadol in rabbit blood and tissues, along with fly larvae, to assess tramadol’s impact on the growth of L. sericata larvae. According to the research, tramadol concentrations in larvae were much lower than those detected in tissues, treated rabbits reached maximum size faster than control rabbits, and the overall developing period rose significantly when the first administered dose was increased.
Malathion was noticed in Ch. albiceps maggots and tissues of treated dog carcasses in a research by Fouda et al. (2017a), where it dramatically changed the durations of developing stages of flies inhabiting the treated carcass but had no effect on the insect number drawn to the carcasses. Fouda et al. (2017b) were also capable to detect ephedrine in Ch. albiceps larvae, and the influence of ephedrine on the growth of maggots. In addition, Tahoun and Abouzied (2017) used GC-Ms analysis to detect tramadol in S. argyrostoma maggots and investigate the influence of tramadol accumulation on maggots and pupae development rates. They observed that tramadol accumulation decreased the maggots and pupae lifespans. When maggots were fed tramadol-treated livers, the average weight of the 3rd instar larvae decreased while the pupae weight rose.
Bakr et al. (2018) looked at the impact of malathion on Ch. megacephala larval development, and the consequences for PMI estimation. Malathion was detected in all treated rabbit tissues, but not in the controls, it was detected in the lungs in the highest intensities, followed by muscle tissue and lipids, as well as in all 3rd larval instars and pupae.
The impacts of morphine on the growth in four flies, L. sericata, Ch. albiceps, Ch. megacephala, and S. argyrostoma, were studied by El-Samad et al. (2020). The drug was found in both blood and samples of tissue taken from treated rabbits, albeit the amounts of morphine found in larval samples for all fly species were lower than in rabbit tissues. Abd El-Gawad et al. (2020) looked at the possibility of using fly larvae as diagnostic indicators for warfarin toxicity. Maggots exhibited two distinct results: positive, which may be owing to the larvae eating on the rabbit liver, and negative, which could be owing to the toxin being highly destroyed after 18 hours of ingestion.
In general, forensic entomotoxicology was applied by numerous Egyptian researchers to check for drugs like tramadol, malathion, morphine, ephedrine, and warfarin in insect larvae and carcass tissues.
Biochemistry is the analysis of biological processes at the cellular and molecular level using chemicals. Sawaby et al. (2009) conducted the only study in Egypt to explore how the pesticide pirimiphos-methyl changes the protein composition of insects drawn to treated rabbit carrion, with the insecticide’s effect on insects. According to the research results, Ch. albiceps and Dermestis maculatus DeGeer contain a functional esterase system. The value of entomological techniques for detecting pharmaceuticals in insects that infested drug-death suspect carcasses, for instance protein profiles and esterase assays, which are involved in physiological and endogenous metabolic processes, is highlighted in this work.
Electrophysiology is a subfield of physiology that studies the electrical properties of biological cells and tissues. There have been several probes of chemosensory sensilla in a variety of insect families, but there is comparatively little information about forensic fly species. Refai et al. (2009) investigated the electrical reactions of receptor nerve cell on the antennae, labium, and tarsus of Calliphora vicina Robineau-Desvoidy to various quantities of morphine in decaying tissues alongside odors originating from under the surface. The 5th cell “deterrent cell” was shown to be the best responsive cell to morphine sulfate at all sites of stimulation, although with varying levels of sensitivity. These findings clearly imply that varying dosages of morphine sulfate have a great repellant impact on C. vicina attraction. Also, to investigate if morphine sulfate has a repellant impact on blowfly attraction to corpses, Abdel Ghaffar et al. (2012) conducted electrophysiological and behavioral studies. They realized that the drug stimulated the fifth cell in the chemosensillae, causing a delay in blowfly attraction to treated carrions compared to control carrions. Researchers concluded that entomologists must be conscious of the delay in adult fly arrival to the carrion in cases where the victim died because of morphine abuse, which could lead to a misinterpretation of PMI estimation, especially when previous studies were showed on morphine-free experimental carcasses.
Human case study
Forensic entomology, according to Hall (1990), concentrates on insect-infested human cadavers. Insects may represent crucial tools in criminal investigations, in addition to their biological significance (Catts, 1992). Insects can be designed to estimate the time since death, especially in the case of putrefied corpses (Singh, A. et al. 2019). Insects can also aid when typical PMI estimation methods based on early postmortem phenomena like body cooling, livor mortis, and rigor mortis fail (Saukko & Knight, 2004). Several experts have studied the insects that are drawn to human remains in different countries (Lee, 1989; Barreto et al., 2002; Lira et al., 2020, Mashaly et al., 2020). Also, no insect attraction to human cases has been studied in the regions around Egypt, except for Saudi Arabia (Al-Qahtni et al., 2019, 2020). In Egypt, a few studies on human cases were done to document the drawn insects to the bodies and attempt to apply this knowledge in estimating the PMI.
Galal et al. (2009) investigated the insect fauna and SPs on degraded human leftover parts in Assiut during the summer and spring. Flesh flies, blowflies, house flies, skin beetles, and ants were all found, with flies being the highly frequent insects and Ch. albiceps being the extremely prevalent species. Moreover, El-Mehy et al. (2015) investigated the ability of insects to estimate PMI in 15 human cases in Gharbia (11 men and 4 females). Nine cases were found in open habitats, three in aquatic environments, and three in subterranean habitats, with ages ranging from 7 to 50 years. In the 15 carcasses, blowflies and flesh fly maggots were found, with Ch. albiceps being the most common (9 instances) and Sarcophaga species following closely behind (4 cases). In two cases killed by carbamate and organophosphorus poisoning, toxicological investigations using larvae attached to decaying remains were helpful. Besides, Tantawi et al. (2018) collected insects during an autopsy of 15 human bodies at El-Esaaf Mortuary in Kom El-Deka, Alexandria. Six fly species maggots from three families were gathered from the remains, as these flies were the first to occupy the remains and hence are crucial in PMI estimating in Alexandria. Blowfly maggots were the highly frequent and numerous insects in all times and environments, with Ch. albiceps being the extremely common species; however, in warmer seasons, S. argyrostoma was revealed to be a very indicative species.
Three studies on human cases were conducted in Egypt, in three distinct governorates, to recording the drawn insects to the dead corpses and PMI estimation.